A Multi-Locus Study for Detection of Cryptosporidium Species Isolated from Calves Population, Liverpool; UK.

Cryptosporidium is an obligate intracellular protozoan parasite infecting a wide range of hosts. The current study investigated the genetic profile of Cryptosporidium species in calves in Liverpool, England. Fifty-two calve fecal samples were collected from a farm and initially screened by Auramine Phenol, modified Ziehl-Neelsen and ELISA. PCR analysis of 18S rRNA gene was carried out for the positive samples. Then, positive PCR samples were genotyped by an 18S rRNA- based PCR-RFLP, COWP - based PCR- RFLP; PCR of GP60 and HSP70 genes. Additionally, sequence analysis was carried out based on representative isolates of four loci. Cryptosporidium oocysts and antigens were detected in 34 out of 52 (65.4%) samples using screening techniques. Genotype analysis showed the presence of C. hominis and C. parvum in one and thirteen samples, respectively. Furthermore, subtypes of C. hominis Ib, C. parvum IIa; C. parvum subtype 2 were identified by GP60 and HSP70 sequences, respectively. These findings indicate the diversity of the molecular characteristics of Cryptosporidium species in calves' isolates. Moreover, referring to the literature; we report two new subtypes of C. parvum IIa and a rare case of C. hominis Ib in calves population.

most prevalent species in cattle and also is the main cause of zoonotic cryptosporidiosis in humans. It has been also found in several hosts including lamb, sheep, goat and so on (3)(4).
One of the genotyping tools which are frequently used in the molecular study of this parasite are PCR and PCR-RFLP of the 18S rRNA gene. This gene is highly polymorphic within the genus and is useful as a target for the identification and differentiation of Cryptosporidium species and genotypes (5)(6). Another molecular tool is PCR and PCR-RFLP of the COWP gene which is a single copy gene encoding a major constituent of the inner layer of the Cryptosporidium oocysts wall protein (7).
Moreover, PCR and sequence analysis of the 60 kDa glycoprotein (GP60) gene has been frequently used for sub-typing of various Cryptosporidium isolates (8). The GP60 locus has the highest resolution as a single marker for sub-typing of C.
parvum isolates because of the existence of nearly one-hundred GP60 sub-genotypes of C. hominis and C. parvum. But this tool does not clearly divide C. hominis and C. parvum into two separate groups (9). Furthermore, heat shock protein 70 kDa (HSP70) gene is a good target for sub-typing and multi-locus study of Cryptosporidium isolates. This gene has a high level of heterogeneity spread over the entire sequence of a variety of Cryptosporidium isolates from human and animal hosts (10).
This approach was conducted to perform a multi-locus study for detection of Cryptosporidium species isolated from calves population using PCR, PCR-RFLP and sequence analysis of the 18S rRNA, COWP, GP60 and HSP70 gene fragments.

DNA sequences analyzing
The PCR products of four genes targets were directly sequenced and were rubbed out from the agarose gel and purified by MicroSpin Columns

The phylogeny of the GP60 gene
The phylogenetic relationships between the GP60 sequences of the Cryptosporidium isolates were assessed with a NJ-tree method using the phylogenetic analysis software Phylogeny (16). The tree was anchored by using C. meleagridis as the out-group as this species showed less similarity to the other species.

Nucleotide sequences accession numbers
Nine sequence PCR samples used in this study have been deposited in the GenBank database under accession no: JX547009,KF533078-79, KF537685-89 and KF577776.

Screening Methods
The prevalence rate of Cryptosporidium  (Table 2).

Molecular analysis of 18S rRNA gene
The 18S rRNA gene fragment was amplified in 14 out of 34 positive samples (   were classified as C. parvum IIa allele group (accession no GQ983359 and JF727795).
hominis Ib allele group with 100% sequence identity with previously published sequence (accession no JF727788). Another isolate (J6) which was successfully amplified and sequenced for the 18S rRNA gene as C. hominis, did not yield any PCR product for sequencing of the GP60 loci.
Phylogenetic analysis of the GP60 gene using of the NJ-tree method showed that C. hominis and C. parvum isolates formed two different clades ( Figure 2). The phylogenetic position of C.  The proportion of C. parvum (92.9%) found in the current study by the PCR-RFLP of 18S rRNA gene was in agreement with those found in other studies (25,27). For instance, in the UK, 93% of un-weaned calves shed oocysts of C.